We propose to demonstrate the strength of combining our engineered tissue-based drug screening approach and a systematically organized mixture-based chemical library. This approach could become an ultimate tool to develop treatments for complex diseases, such as cardiac fibrosis, without known drug targets initially. Heart failure is the most common cause of hospitalization among Americans 65 or older. The heart failure with preserved ejection fraction (HFPEF) is becoming an epidemic among increasing population of aging Americans. Existence of activated fibroblasts, i.e. myofibroblasts, differentiated from fibroblasts, endothelial cells, and non-muscle cell is a hallmark of myocardium with HFPEF, and they stiffen myocardium to progress HFPEF. We developed a disease model for the human cardiac fibrosis using myofibroblast-containing engineered tissue constructs that recapitulate connective tissues of myocardium with fibrosis. Using our phenotypic screening system, we identified TPI-2049 (17,340 compound mixture) after analyzing a 42-scaffold scaffold ranking library (each scaffold sample contains mixtures of compounds), which is equivalent to screening >30 million compounds. While our final goal is to identify a novel drug for treating cardiac fibrosis, the proposed Phase I project will focus on performing a secondary sample screening of a compound mixture in the chemical scaffolds and identifying 50 individual compounds to pick 15 hit compounds to be tested and optimized in Phase II study. In a parallel study, we will analyze mechanisms of action of TPI-2049 in fibrosis relieving phenotypes and identify potential drug targets by performing affinity-free target identification method, stabilizing target molecules with individual hit compounds. Successful demonstration of combing engineered tissue based drug discovery system and scaffold ranking library with positional scanning technology will be extended to other drug discovery projects for age-related dieses such as osteoarthritis.